Ultrasensitive ctDNA monitoring predicts early response of immunotherapy in recurrent metastatic non-small cell lung cancer

Background: Circulating tumor DNA (ctDNA) offers a minimally invasive approach for response monitoring of cancer immunotherapy treatment and early prediction of therapeutic outcomes. However, the clinical utility of ctDNA-based liquid biopsy faces a critical challenge: reliable ctDNA detection in low-shedding tumors and in patients with low molecular residual disease (MRD) following treatment response. We employed an ultrasensitive ctDNA assay to address this technical limitation, enabling precise longitudinal monitoring essential for optimizing IO therapy. Methods: We analyzed longitudinal plasma samples from 41 patients with non-small cell lung cancer (NSCLC) [adenocarcinoma (n=19), non-adenocarcinoma (n=22)], who received IO monotherapy (n=33) or combined IO and chemotherapy (n=8) in the Deciphering Anti-tumour Response and Resistance With INtratumour Heterogeneity (DARWIN 2) trial nested within the TRACERx study. Using NeXT Personal®, an ultra-sensitive personalized liquid biopsy approach, we tracked up to ~1,800 patient-specific somatic variants per case across 233 plasma samples. Results: The median limit of detection across all tests was 1.52 parts per million (PPM), enabling ctDNA detection across six orders of magnitude (range 2.1-309,673 PPM), with 21% of positive ctDNA detections in the ultrasensitive range (<100 PPM), with that increasing to 28% while on treatment. Histological subtype had no impact on detection with this assay. Early molecular response (mR), defined as either >50% reduction in ctDNA or sustained ctDNA negativity from pre-treatment baseline to the subsequent plasma sample (median interval: 43.5 days), was significantly associated with improved clinical outcomes. Patients achieving early mR exhibited superior progression-free survival (PFS; HR = 0.33, 95% CI 0.14-0.77, p = 0.010) and overall survival (OS; HR = 0.31, 95% CI 0.14-0.70, p = 0.005). All patients with complete response by RECIST criteria achieved early mR (sensitivity = 100%). Conversely, disease in all patients lacking mR progressed within 15 months. Furthermore, durable molecular complete response (dmCR), defined as ctDNA negativity maintained for ≥180 days, was strongly associated with improved survival outcomes (2-year PFS: 50% vs. 9%, HR = 0.31 95%CI 0.11-0.90, p = 0.032; 2-year OS: 86% vs. 13%, HR = 0.06, 95% CI 0.01-0.48, p = 0.007 for dmCR vs. non-dmCR, respectively). Conclusions: Early ctDNA kinetics serve as a robust predictor of long-term immunotherapy outcomes in patients with advanced NSCLC. The ability to detect ultra-low ctDNA levels allowed for accurate assessment of minimal residual disease, irrespective of lung cancer histology. These findings establish ultrasensitive ctDNA monitoring as a valuable tool for precise, real-time evaluation of immunotherapy response, with implications for clinical decision-making.